The present invention provides new psoralens and methods of synthesis of new psoralens having enhanced ability to inactivate pathogens in blood products in the presence of ultraviolet light, without significantly effecting blood product function or exhibiting mutagenicity.
Pathogen contamination within the blood supply remains an important medical problem throughout the world. Although improved testing methods for hepatitis B (HBV), hepatitis C (HCV), and HIV have markedly reduced the incidence of transfusion associated diseases, the public is losing trust in the safety of the blood supply due to publicity of cases of transfusion related transmission of these viruses.
For example, the recent introduction of a blood test for HCV will reduce transmission of this virus; however, it has a sensitivity of only 67% for detection of probable infectious blood units. HCV is responsible for 90% of transfusion associated hepatitis. Melnick, J. L., abstracts of Virological Safety Aspects of Plasma, Cannes, Nov. 3-6 (1992) (page 9). It is estimated that, with the test in place, the risk of infection is 1 out of 3300 units transfused.
Similarly, while more sensitive seriological assays are in place for HIV-1 and HBV, these agents can nonetheless be transmitted by seronegative blood donors. International Forum: Vox Sang 32:346 (1977). Ward, J. W., et al., N. Engl. J. Med., 318:473 (1988). Up to 10% of total transfusion-related hepatitis and 25% of severe icteric cases are due to the HBV transmitted by hepatitis B surface antigen (HBasAg) negative donors. To date, fifteen cases of transfusion-associated HIV infections have been reported by the Center for Disease Control (CDC) among recipients of blood pre tested negative for antibody to HIV-1.
Furthermore, other viral, bacterial, and agents are not routinely tested for, and remain a potential threat to transfusion safety. Schmunis, G. A., Transfusion 31:547-557 (1992). In addition, testing will not insure the safety of the blood supply against future unknown pathogens that may enter the donor population resulting in transfusion associated transmission before sensitive tests can be implemented.
Even if seroconversion tests were a sufficient screen, they may not be practical in application. For example, CMV (a herpes virus) and parvo B19 virus in humans are common. When they occur in healthy, immunocompetent adults, they nearly always result in asymptomatic seroconversion. Because such a large part of the population is seropositive, exclusion of positive units would result in substantial limitation of the blood supply.
An alternative approach to eliminate transmission of viral diseases through blood products is to develop a means to inactivate pathogens in transfusion products. Development of an effective technology to inactivate infectious pathogens in blood products offers the potential to improve the safety of the blood supply, and perhaps to slow the introduction of new tests, such as the recently introduced HIV-2 test, for low frequency pathogens. Ultimately, decontamination technology could significantly reduce the cost of blood products and increase the availability of scarce blood products.
To be useful, such an inactivation method i) must not adversely effect the function for which the blood product is transfused, ii) must thoroughly inactivate existing pathogens in the blood product, and iii) must not adversely effect the recipients of the blood product. Several methods have been reported for the inactivation or elimination of viral agents in erythrocyte-free blood products. However, most of these techniques are completely incompatible with maintenance of the function of platelets, an important blood product. Examples of these techniques are: heat (Hilfenhous, J., et al., J. Biol. Std. 70:589 (1987)), solvent/detergent treatment (Horowitz, B., et al., Transfusion 25:516 (1985)), gamma-irradiation (Moroff, G., et al., Transfusion 26:453 (1986)), UV radiation combined with beta propriolactone, (Prince A. M., et al., Reviews of Infect. Diseases 5: 92-107 (1983)), visible laser light in combination with hematoporphyrins (Matthews J. L., et al., Transfusion 28: 81-83 (1988); North J., et al., Transfusion 32: 121-128 (1992)), use of the photoactive dyes aluminum phthalocyananine and merocyanine 540 (Sieber F., et al., Blood 73: 345-350 (1989); Rywkin S., et al., Blood 78(Suppl 1): 352a (Abstract) (1991)) or UV alone (Proudouz, K. N., et al., Blood 70:589 (1987)).
Other methods inactivate viral agents by treatment with furocoumarins, such as psoralens, in the presence of ultra-violet light. Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA). G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985); Hearst et al., Quart. Rev. Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to a psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a second photon can lead to formation of a diadduct which functions as an interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058 (1977); S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982); J. Tessman et al., Biochem. 24:1669 (1985); Hearst et al., U.S. Pat. Nos. 4,124,598, 4,169,204, and 4,196,281, hereby incorporated by reference.
The covalently bonded psoralens act as inhibitors of DNA replication and thus have the potential to stop the replication process. Due to this DNA binding capability, psoralens are of particular interest in relation to solving the problems inherent in creating and maintaining a pathogen blood supply. Some known psoralens have been shown to inactivate viruses in some blood products. H. J. Alter et al., The Lancet (ii:1446) (1988); L. Lin et al., Blood 74:517 (1989) (decontaminating platelet concentrates); G. P. Wiesehahn et al., U.S. Pat. Nos. 4,727,027 and 4,748,120, hereby incorporated by reference, describe the use of a combination of 8-methoxypsoralen (8-MOP) and irradiation. P. Morel et al., Blood Cells 18:27 (1992) show that 300 xcexcg/mL of 8-MOP together with ten hours of irradiation with ultraviolet light can effectively inactivate viruses in human serum. Similar studies using 8-MOP and aminomethyltrimethyl psoralen (AMT) have been reported by other investigators. Dodd R Y, et al., Transfusion 31:483-490 (1991); Margolis-Nunno, H., et al., Thromb Haemostas 65:1162 (Abstract)(1991). Indeed, the photoinactivation of a broad spectrum of microorganisms has been established, including HBV, HCV, and HIV, under conditions different from those used in the present invention and using previously known psoralen derivatives. [Hanson, C. V., Blood Cells: 18: 7-24 (1992); Alter, H. J., et al., The Lancet ii:1446 (1988); Margolis-Nunno, H. et al., Thromb Haemostas 65: 1162 (Abstract) (1991).]
Psoralen photoinactivation is only feasible if the ability of the psoralen to inactivate viruses is sufficient to ensure a safety margin in which complete inactivation will occur. On the other hand, the psoralen must not be such that it will cause damage to blood products. The methods just described, when applied using known psoralens, require the use of difficult and expensive procedures to avoid causing damage to blood products. For example, some compounds and protocols have necessitated the removal of molecular oxygen from the reaction before exposure to light, to prevent damage to blood products from oxygen radicals produced during irradiation. See L. Lin et al., Blood 74:517 (1989); U.S. Pat. No. 4,727,027, to Wiesehahn. This is a costly and time consuming procedure.
Finally, some commonly known compounds used in photochemical decontamination (PCD) exhibit undesirable mutagenicity which appears to increase with increased ability to kill virus. In other words, the more effective the known compounds are at inactivating viruses, the more injurious the compounds are to the recipient, and thus, the less useful they are at any point in an inactivation system of products for in vivo use.
A new psoralen compound is needed which displays improved ability to inactivate pathogens and low mutagenicity, without causing significant damage to blood products for which it is used, and without the need to remove oxygen, thereby ensuring safe and complete inactivation of pathogens in blood decontamination methods.
The present invention provides new psoralens and methods of synthesis of new psoralens having enhanced ability to inactivate pathogens in the presence of ultraviolet light which is not linked to mutagenicity. The present invention also provides methods of using new and known compounds to inactivate pathogens in health related products to be used in vivo and in vitro, and particularly, in blood products and blood products in synthetic media.
The present invention contemplates a method of inactivating pathogens in a platelet preparation comprising, in the following order: a) providing, in any order, i) a synthetic media comprising a compound selected from the group consisting of 4xe2x80x2-primaryamino-substituted psoralens and 5xe2x80x2-primaryamino-substituted psoralens; ii) photoactivating means for photoactivating said compound; and iii) a platelet preparation suspected of being contaminated with a pathogen having nucleic acid; b) adding said synthetic media to said platelet preparation; and c) photoactivating said compound so as to prevent the replication of substantially all of said pathogen nucleic acid, without significantly altering the biological activity of said platelet preparation. The pathogen may be a virus, or a bacteria. Its nucleic acid may be single stranded or double stranded, DNA or RNA. The photoactivating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm. The intensity may be between 1 and 30 mW/cm2 and the platelet preparation is exposed to said intensity for between 1 second and thirty minutes. The spectrum of electromagnetic radiation may be wavelengths between 320 nm and 380 nm.
In one embodiment the compound displays low mutagenicity. It may be added to said platelet preparation at a concentration of between 0.1 and 250 xcexcM. And the method may be performed without limiting the concentration of molecular oxygen.
The 4xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 4xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)2xe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NHz, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents R5, R6, and R7 on the 4, 5xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
Alternatively, the 5xe2x80x2-primaryamino-substituted psoralen comprises: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4, 4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5, and where when R1 is selected from the group comprising xe2x80x94(CH2)uxe2x80x94NH2, R6 is H; or a salt thereof.
Finally, the 5xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 3 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4,4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
In one embodiment, at least two compounds are present. In another embodiment, the synthetic media further comprises sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride, and may also include mannitol and/or glucose.
In one embodiment, the synthetic media is contained in a first blood bag and said platelet preparation is contained in a second blood bag, the synthetic media being added to the platelet preparation in step (b) by expressing the synthetic media from the first blood bag into the second blood bag via a sterile connection.
In a preferred embodiment, the compound is either 5xe2x80x2-(4-amino-2-oxa)butyl-4,4xe2x80x2,8-trimethylpsoralen or 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
In one embodiment, the method described above includes administering said platelet preparation by intravenous infusion to a mammal.
In another embodiment, method of inactivating pathogens in a platelet preparation comprising, in the following order: a) providing, in any order, i) a synthetic media comprising a buffered saline solution and a compound displaying low mutagenicity, selected from the group consisting of 4xe2x80x2-primaryamino-substituted psoralens and 5xe2x80x2-primaryamino-substituted psoralens, contained in a first blood bag; ii) photoactivating means for photoactivating said compound; and iii) a platelet preparation suspected of being contaminated with a pathogen having nucleic acid, contained in a second blood bag; b) adding said synthetic media to said platelet preparation by expressing said synthetic media from said first blood bag into said second blood bag via sterile connection means; and c) photoactivating said compound so as to prevent the replication of substantially all of said pathogen nucleic acid, without significantly altering the biological activity of said platelet preparation. The pathogen may be a virus, or a bacteria. Its nucleic acid may be single stranded or double stranded, DNA or RNA. The photoactivating means comprises a photoactivation device capable of emitting a given intensity of a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm. The intensity may be between 1 and 30 mW/cm2 and the platelet preparation is exposed to said intensity for between 1 second and thirty minutes. The spectrum of electromagnetic radiation may be wavelengths between 320 nm and 380 nm.
In one embodiment the compound displays low mutagenicity. It may be added to said platelet preparation at a concentration of between 0.1 and 250 xcexcM. And the method may be performed without limiting the concentration of molecular oxygen.
The 4xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 4xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents R5, R6, and R7 on the 4,5xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
Alternatively, the 5xe2x80x2-primaryamino-substituted psoralen comprises: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4,4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5, and where when R1 is selected from the group comprising xe2x80x94(CH2)uxe2x80x94NH2, R6 is H; or a salt thereof.
Finally, the 5xe2x80x2-primaryamino-substituted psoralen may comprise: a) a substituent R1 on the 5xe2x80x2 carbon atom, selected from the group comprising:
xe2x80x94(CH2)uxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)zxe2x80x94NH2,
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)zxe2x80x94NH2, and
xe2x80x94(CH2)wxe2x80x94R2xe2x80x94(CH2)xxe2x80x94R3xe2x80x94(CH2)yxe2x80x94R4xe2x80x94(CH2)zxe2x80x94NH2;
wherein R2, R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 3 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R6, and R7 on the 4,4xe2x80x2, and 8 carbon atoms respectively, independently selected from the group comprising H and (CH2)vCH3, where v is a whole number from 0 to 5; or a salt thereof.
In one embodiment, at least two compounds are present. In another embodiment, the synthetic media further comprises sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride, and may also include mannitol and/or glucose.
In one embodiment, the synthetic media is contained in a first blood bag and said platelet preparation is contained in a second blood bag, the synthetic media being added to the platelet preparation in step (b) by expressing the synthetic media from the first blood bag into the second blood bag via a sterile connection.
In a preferred embodiment, the compound is either 5xe2x80x2-(4-amino-2-oxa)butyl-4,4xe2x80x2,8-trimethylpsoralen or 4xe2x80x2-(4-amino-2-oxa)butyl-4,5xe2x80x2,8-trimethylpsoralen.
In one embodiment, the method described above includes administering said platelet preparation by intravenous infusion to a mammal.
The present inventions also contemplates a method of synthesizing 4,8-dialkyl-5xe2x80x2-bromomethyl-4xe2x80x2-methylpsoralens, without performing chloromethylation, comprising: a) providing 4,8-dialky-7-(1-methyl-2-oxopropyloxy)psoralen; d) stirring 4,8-dialky-4xe2x80x2, 5xe2x80x2-dimethylpsoralen in carbon tetrachloride to obtain 4,8-dialkyl-5xe2x80x2-bromomethyl-4xe2x80x2-methylpsoralen. A method of synthesizing 4,8-dialkyl-4xe2x80x2-bromomethyl-5xe2x80x2-methylpsoralens , without performing chloromethylation, is contemplated, comprising: a) providing 4,8-dialky-7-(1-methyl-2-oxopropyloxy)psoralen; d) stirring 4,8-dialky-4xe2x80x2, 5xe2x80x2-dimethylpsoralen in methylene chloride to obtain 4,8-dialkyl-4xe2x80x2-bromomethyl-5xe2x80x2-methylpsoralen.
A novel compound is also contemplated, having the formula: 
or a salt thereof.
Finally, the present invention contemplates compositions having anti-viral properties. The first comprising an aqueous solution of a 4xe2x80x2-primaryamino-substituted psoralen and platelets suitable for in vivo use. One embodiment, further comprises a synthetic media, comprising sodium acetate, potassium chloride, sodium chloride, sodium citrate, sodium phosphate and magnesium chloride and optionally mannitol or glucose. These same compositions are contemplated that contain a 5xe2x80x2-primaryamino-substituted psoralen rather than a 4xe2x80x2-primaryamino-substituted psoralen.
A novel synthetic platelet storage media, is also contemplated, comprising an aqueous solution of:
45-100 mM sodium chloride;
4-5 mM potassium chloride;
10-15 mM sodium citrate;
20-27 mM sodium acetate;
0-2 mM glucose;
0-30 mM mannitol;
approximately 20 mM sodium phosphate;
2-3 mM magnesium chloride; and
a psoralen selected from the group consisting of 4xe2x80x2-primaryaminopsoralen and a 5xe2x80x2-primaryaminopsoralen, at a concentration between approximately 0.1 and 250 xcexcM.